Apelin (APLN), the endogenous mammalian peptide ligand of the apelin receptor has been indicated as a regulator of the cardiovascular system. Human apelin is a pre-proprotein of 77 amino acids (MNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPW QGGRRKFRRQRPRLSHKGPMPF (SEQ ID NO: 6)), with a secretory signal sequence in the N-terminal region. After cleavage of the signal peptide at the endoplasmic reticulum, the remaining 55 amino acid residue may undergo further cleavage to several active isoforms including a 36 amino acid peptide corresponding to apelin sequence residues 42-77 (apelin-36, LVQPRGSRNGPGPWQGGRRKFRR-QRPRLSHKGPMPF, (SEQ ID NO:3)) (3), a 17 amino acid peptide corresponding to the apelin sequence residues 61-77 (apelin-17, KFRRQRPRLSHKGPMPF (SEQ ID NO:2)) (2) and a 13 amino acid peptide corresponding to the apelin sequence residues 65-77 (apelin-13, QRPRLSHKGPMPF (SEQ ID NO:7)), which all possess a conserved C-terminal amino acid (FIG. 1). The apelin-13 fragment may also undergo subsequent pyroglutamylation at its N-terminal glutamine residue to provide (pyr1) apelin-13 ((Pyr)RPRLSHKGPMPF (SEQ ID NO:1) (1).
Apelin pathway mediates a positive effect on cardiac contractility and vasodilator activity that counteracts angiotensin-II-induced vasoconstriction. Moreover, apelin administration has been indicated to reduce the progression of cardiac hypertrophy, while apelin knockout mice have been shown to be susceptible to heart failure. Apelin also has a beneficial role in the cardiovascular system, such as initiating vasodilation through a NO-mediated mechanism, positive inotropy, angiogenesis, and the prevention of myocardial ischemic reperfusion injury.
Despite the beneficial physiological effects of the apelinergic system, the lifespan of apelin peptides is heavily regulated and limited via proteolysis. The (pyr1) apelin-13 fragment (SEQ ID NO:1), for example, has been indicated as an endogenous ligand for the apelin receptor with an EC50 about 0.37 nM, while (pyr1) apelin-13 exhibits potent vascular effects in vivo. However, (pyr1) apelin-13 stability is quite low in human plasma, with a t1/2 of about one minute.
Angiotensin converting enzyme 2 (ACE2) is a well-known monocarboxypeptidase that efficiently catalyzes the removal of the conserved C-terminal phenylalanine from apelin isoforms in vitro and in vivo. Des-phenylalanine apelin isoforms behave as biased agonists by retaining native binding and forskolin-induced cAMP inhibition, but abolishing apelin receptor internalization and β-arrestin recruitment. Studies have shown that truncated peptides demonstrate a diminished capacity to lower blood pressure and have no ability to protect against myocardial ischemic reperfusion injury, making the C-terminal Phe residue essential for full agonist activity. Therefore, there exists a need to negate the impact of ACE2 degradation on apelin isoforms, and to improve the overall stability of the apelin isoforms. Given the therapeutic potential of (pyr1) apelin-13 and related apelin peptides, there is a continued interest in stabilizing the peptide structures while preserving their biological profiles.